Gene silencing or inhibiting the expression of a gene holds great therapeutic and diagnostic promise. An example of this approach is antisense technology which can be used to inhibit gene expression in viva. However, many problems remain with development of effective antisense technology. For example, DNA antisense oligonucleotides exhibit only short term effectiveness and are usually toxic at the doses required. Similarly, the use of antisense RNAs has also proved ineffective due to stability problems.
Other approaches to quelling specific gene activities are posttranscriptional gene silencing (PTGS) and RNA interference (RNAi) phenomena, which have been found capable of suppressing gene activities in a variety of in-vivo systems, including plants (Grant, S. R. (1999) Cell 96, 303-306), Drosophila melanogaster (Kennerdell, J. R. and Carthew, R. M. (1998) Cell 95, 1017-1026, Misquitta, L. and Paterson, B. M. (1999) Proc. Natl. Acad. Sci. USA 96, 1451-1456, and Pal-Bhadra, M., Bhadra, U., and Birchler, J. A. (1999) Cell 99, 35-46), Caenorhabditis elegans (Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., and Timmons, L. (1999) Cell 99, 123-132, Ketting, R. F., Haverkamp, T. H., van Luenen, H. G., and Plasterk, R. H. (1999) Cell 99, 133-141, Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Nature 391, 806-811 and Grishok, A., Tabara, H., and Mello, C. C. (2000) Science 287, 2494-2497), zebrafish (Wargelius, A., Ellingsen, S., and Fjose, A. (1999) Biochem. Biophys. Res. Commun. 263, 156-161) and mouse (Wianny, F. and Zernicka-Goetz, M. (2000) Nature Cell Biol. 2, 70-75). In general, the transfection of a plasmid-like DNA structure (transgene) into cells induces PTGS phenomena, while that of a double-stranded RNA (ds-RNA) causes an RNAi effect.
These phenomena appear to evoke an intracellular sequence-specific RNA degradation process, affecting all highly homologous transcripts, called cosuppression. It has been proposed that such cosuppression results from the generation of small RNA products (21˜25 nucleotide bases) by an RNA-directed RNA polymerase (RdRp) (Grant supra) and/or a ribonuclease (RNase) (Ketting et al. supra, Bosher, J. M. and Labouesse, M. (2000) Nature Cell Biology 2, 31-36 and Zamore, P. D., Tuschl, T., Sharp, P. A., and Bartel, D. P. (2000) Cell 101, 25-33.) activity on an aberrant RNA template, derived from the transfecting nucleic acids or viral infection. Although an RdRp-independent endoribonucleolysis model has been proposed for the RNAi effect in Drosophila (Zamore, et al. supra), the RdRp homologues were widely found in Arabidopsi thalianas as Sde-1/Sgs-2 (Yang, D., Lu, H., and Erickson, J. W. (2000) Current Biology 10, 1191-1200), Neurospora crassa as Qde-1 (Cogoni, C. and Macino, G. (1999) Nature 399, 166-169) and Caenorhabditis elegans as Ego-1 (Smardon, A., Spoerke, J. M., Stacey, S. C., Klein, M. E., Mackin, N., and Maine, E. M. (2000) Curr. Biol. 10, 169-171). Thus, RdRp homologues appear to be a prerequisite for maintaining a long-term/inheritable PTGS/RNAi effect (Basher, et al. supra).
Although PTGS/RNAi phenomena appear to offer a potential avenue for inhibiting gene expression, they have not been demonstrated to work well in higher vertebrates and, therefore, their widespread use in higher vertebrates is still questionable. Consequently, there remains a need for an effective and sustained method and composition for inhibiting gene function in vivo in higher vertebrates.